The rapid and successful transmission of electrical signals in myelinated nerve fibers requires axoglial interaction and the proper subcellular distribution of ion channels. This fact is well illustrated by the high degree of regulation in Na+ channel localization, clustering, and isoform expression at nodes of Ranvier. Recent work suggests that this regulation is dependent on both myelin and specific neuroglial interactions at the paranode, the site where sequential layers of the myelin sheath terminate against the axon in septate like axoglial junctions. Diseases or injuries that result in demyelination (such as multiple sclerosis and spinal cord injury) result in conduction block not only through changes to the passive electrical properties of the nerve fiber, but also because the precise localization and clustering of ion channels is disrupted. This proposal aims to test the hypothesis that neuroglial interactions regulate the expression, clustering, and localization of Na+ channels at nodes of Ranvier. To do this, we will: 1. Identify and characterize new molecular components of the node and paranode by immunoaffinity purification of nodal and paranodal protein complexes; these purified proteins will be characterized by mass-spectrometry. Bona fide interactions with known proteins will be verified by co-immunoprecipitation and colocalization in native tissueand transfected cells. 2. Identify and characterize the antigens for a library of monoclonal antibodies wehave made against unknown nodal and paranodal proteins. The functions of newly identified antigens will be determined through a developmental analysis of expression at nodes of Ranvier, and by coimmunoprecipitation and colocalization with known proteins from/in brain and spinal cord. 3. Identify the function of the myelin sheath and paranode in regulating Na+ channel isoform expression at nodes of Ranvier by examining expression during chronic and acute demyelination, and in mutant mice with altered paranodal structure.